Primer Walking (SS1008, SS1009)

We provide single directional and bi-directional primer walking of DNA constructs.

Bi-directional primer walking is recommended for more accurate results.

1) Bi-directional Primer Walk

For bi-directional primer walking, DNA sequencing will start with two universal primers and is continued using primers derived from the ends of the newly determined DNA sequences until the whole insert/ fragment is fully sequenced until both ends. If there are any ambiguities in the sequence, we will design new primers to resolve them.

Figure 1: Bi-directional primer walking of construct provides 99.9% accuracy with a double confirmation of the insert sequence on both strands.

2) Single Pass Primer Walk

For single pass primer walking, DNA sequencing will start with two universal primers and is continued using primers derived from the ends of the newly determined DNA sequences until the overlapping region of both strands are determined.

Figure 2: Single pass primer walking of constructs provides a faster turnaround rate with 98.5% accuracy of the insert sequence.

Primer Walking Service Includes:

  • Primer design & synthesis
  • DNA sequencing reactions using the designed primer(s)
  • Data assembly and sequence aligning to generate the consensus sequence

Specifications

Below are the sample requirements: -

Length Concentration & Volume/ Project
Primer Walking [<= 3kb] 100 ng/µL, min 50 µL per rxn in dH2O 1.5kb per week
Primer Walking [> 3kb] Whole bacterial colony OR 100 ng/µL, min 50 µL per rxn in dH2O

The service is charged as per length of cloned insert, and is only recommended for insert sizes less than 7 kbp. Please contact your Local Sales Office if you have an insert > 7 kbp. In such instances we may process your order as Molecular Biology Services.

The base pair charges start from the first until the last base of the inserted fragment in the holding vector. As such, customer is required to provide vector name and RE cut sites (if this applies) for data alignment. This is provided the 1st round of reactions using the Universal Primers of the holding vector are successful, otherwise it will be charged as regular single pass sequencing reaction (SS1001).